Polyamines have demonstrated many useful biological properties and are under study as active pharmaceutical agents for many medical conditions. See, e.g., Senanayake T. et al. Essay Biochem., 46:77-94 (2013); Zini M. et al. Chemico-Biological Interactions, 181:409-416 (2009); Kaur N. et al. J. Med. Chem., 51:2551-2560 (2008); Boncher, T. et al. Biochem. Soc. Trans., 35(2):356-363 (2007); Melchiorre C. et al. J. Med. Chem., 53:5906-5914 (2010); and Polyamine Drug Discovery, edited by Patrick Woster and Robert Casero, RCS Publishing, 2011, DOI:10.1039/9781849733090.
For example, certain polyamines have been identified as inhibitors of monoamine oxidase A and B (MAO A and MAO B) and vascular adhesion protein 1 (VAP-1), suggesting they may be useful in anti-neurodegenerative and anti-depressant therapies such as Parkinson's and Alzheimer's diseases, and affective disorders. See, e.g., Bonaiuto E. et al., Eur. J. Med. Chem., 70:88-101 (2013). For other reports of the neuroprotective effects of polyamines and/or their use in treating mental and neurological disorders, see, e.g., Zhang X. et al. Acta Pharmaceutica Sinica B, 5(1):67-73 (2015); Saiki R. et al. Bioorganic & Medicinal Chem. Letters, 23:3901-3904 (2013); Fiori L M et al. J. Psychiatry Neurosci., 33(2):102-110 (2008); and Gilad G M and Gilad V H, J. Pharmacology and Experimental Therapeutics, 291(1):39-43 (1999).
Cancer chemotherapy and chemoprevention is another utility for polyamine pharmaceuticals. See, e.g., Murray-Stewart T. et al. Amino Acids, 46(3):585-594 (2014); Casero R A, Cancer Discovery, 975-977 (September 2013); Minarini A. et al. European J. Medicinal Chem., 67:359-366 (2013); Casero R A and Woster P M, J. Med. Chem., 52:4551-4573 (2009); Rossi T. et al. Anticancer Research, 28:2765-2768 (2008); Seiler N. and Raul F. J. Cell. Mol. Med. 9(3):623-642 (2005).
Polyamines are also under investigation as treatment for tropical diseases. See, e.g., Verlinden B K et al. Bioorganic & Medicinal Chemistry, 23:5131-5143 (2015); and O'Sullivan M C et al. Bioorganic & Medicinal Chemistry, 23:996-1010 (2015).
The immunomodulatory effect of increased polyamine metabolism has been detailed in many scientific reports. Several studies have demonstrated an immunological inhibitory effect of increased levels of polyamines surrounding tumors. For example, Moulinoux and coworkers described experiments where a complete depletion of polyamine levels in mice grafted with 3LL (Lewis lung) carcinoma was accomplished by treatment with DFMO, a polyamine oxidase inhibitor and neomycin to prevent the gut microbial flora from providing polyamines. In these mice, tumor growth was reduced and immune system abnormalities seen in tumor-bearing animals were reversed. See, e.g., Chamaillard, L, et al. Polyamine deprivation prevents the development of tumor-induced immune suppression. British Journal of Cancer, 76:365-370 (1997). The decreased spleen cell interleukin 2 (IL-2) production and CD4+ and CD8+ lymphocyte populations observed prior to treatment with drugs were reversed and previously increased polyamine levels in the spleen were lowered. It was necessary to maintain a total blockage of all major polyamine sources to see these reversals. The T-lymphocyte population restoration did not depend upon the stage of tumor growth. No other vaccine activation or tumor-directing antigens were required.
Additionally, Moulinoux and coworkers examined the effects of more total polyamine depletion in mice grafted with 3LL carcinoma in relation to the re-stimulation of the non-specific immune system specializing in tumor cell killing. See, e.g., Chamaillard, L., et al. Polyamine deprivation stimulates natural killer cell activity in cancerous mice. Anticancer Research, 13:1027-1033 (1993). The decrease in the cytotoxic activity of the mouse's natural killer (NK) cells was reversed in these polyamine depleted animals. The authors conclude that polyamines, secreted by the tumor itself as well as absorbed through the gastrointestinal tract, can be considered not only as autocrine growth factors but also as natural immunosuppressive factors.
Soda and coworkers studied the effects of polyamines on cellular immune function. See, e.g., Kano, Y., et al. Increased blood spermine levels decrease the cytotoxic activity of lymphokine-activated killer cells: a novel mechanism of cancer evasion, Cancer Immunology, Immunotherapy, 56:771-781 (2007). Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured with spermine, spermidine or putrescine and the results on immune cell function were examined. Treatment resulted in decreased adhesion of non-stimulated PBMCs to tissue culture plastic in a dose- and time-dependent manner without affecting cell viability or activity. This decreased adhesion was also associated with a decrease in the number of CD11a positive and CD56 positive cells. In a group of 25 cancer patients, changes in blood spermine levels after surgery were negatively correlated with changes in lymphokine-activated killer cells (LAK) cytotoxicity. These authors concluded that increased blood spermine levels maybe an important factor in the suppression of antitumor immune cell function.
A study reported by Bowlin noted the effect of the polyamine biosynthesis inhibitor DFMO on immune system cell expression in normal and tumor-bearing (B16 melanoma) C57BL/6 mice. See, e.g., Bowlin, T. L, et al. Effect of polyamine depletion in vivo by DL-alpha-difluoromethylornithine on functionally distinct populations of tumoricidal effector cells in normal and tumor-bearing mice. Cancer Research, 46:5494-5498 (1986). They observed that DFMO treatment of these immune competent mice for 6 days reduced splenic leukocyte polyamine levels and resulted in the induction of cytotoxic T-lymphocytes in both normal and tumor-bearing animals. While putrescine and spermidine levels were significantly reduced, spermine levels were not. This led the authors to suggest that the generation of CTLs is sensitive to spermine levels.
Another study by the same authors explored the effect of treatment by each of three different ornithine decarboxylase inhibitors on tumoricidal macrophage activities in vivo. See, e.g., Bowlin, T. L, et al. Effects of three irreversible inhibitors of ornithine decarboxylase on macrophage-mediated tumoricidal activity and antitumor activity in B16F1 tumor-bearing mice. Cancer Research 50:4510-4514 (1990). Tumor-bearing mice that were treated with 0.5 to 2.0% oral DFMO had two-fold augmented macrophage mediated cytolysis of B16F1 cells ex vivo. An earlier study by Bowlin showed that polyamine oxidation down-regulates IL-2 production by human peripheral blood mononuclear cells. See, e.g., Flescher, E., et al. Polyamine oxidation down-regulates IL-2 production by human peripheral blood mononuclear cells. Journal of Immunology, 142:907-912 (1989).
Gensler reported studies exploring the ability of DFMO to prevent skin carcinogenesis and immunosuppression induced by ultraviolet irradiation in immuno-competent BALB/c mice. Gensler, H. L. Prevention by alpha-difluoromethylornithine of skin carcinogenesis and immunosuppression induced by ultraviolet irradiation. Journal of Cancer Research and Clinical Oncology 117:345-350 (1991). Mice pretreated for 3 weeks with 1% DFMO in their drinking water and then irradiated with UVB radiation had a reduced, 9% occurrence of skin cancer whereas the untreated control group developed cancers in 38% of the mice. The degree of removal of immunosuppression in the DFMO-treated mice was measured by a passive-transfer assay. Splenocytes from UV-irradiated mice when transferred to naïve mice prevented their normal ability to reject UV-induced tumor challenges (20 of 24 of mice grew tumors). When the splenocytes from UV-irradiated mice that where treated with DFMO were transferred to naïve mice, the majority of tumors were rejected (only 2 of 24 grew).
Gervais reported experiments looking at the phenotype and functional activity of dendritic cells from cancer patients and investigated the effect of putrescine on these immune cells. See, e.g., Gervais, A., et al. Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency. Breast Cancer Res., 7:R326-335 (2005). Cells from cancer patients yielded a lower yield of dendritic cells and these cells showed a weaker expression of MHC class II molecules. By adding putrescine to dendritic cells from normal donors, it was possible to reduce the final cytolytic activity of lymphocytes, mimicking the defective dendritic cell function of cancer patients.
Evans observed that spermine suppresses the sensitivity of cervical carcinoma cells to cytotoxic LAK lymphocytes collected from more than half the human subjects studied. See, e.g., Evans, et al. Spermine-directed immunosuppression of cervical carcinoma cell sensitivity to a majority of lymphokine-activated killer lymphocyte cytotoxicity. Nat. Immun., 14:157-163 (1995).
Tracey has reported that spermine has an immune inhibitory effect. See, e.g., Zhang, M., et al. Spermine inhibits pro-inflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response. J Exp. Med., 185:1759-1768 (1997). Specifically, Tracey observed that LPS stimulation of monocytes causes an increase in the uptake of spermine by the polyamine transport apparatus of the cell. They used a polyamine transport inhibitor, 4-bis(3-aminopropyl)-piperazine (BAP) to block the inhibitory activity of spermine on monocyte TNF production.
Experiments using carrageenan-induced inflammation in rats showed BAP enhanced the production of TNFα and increased the resulting edema in the foot pad. See, e.g., Zhang, M., et al. Spermine inhibition of monocyte activation and inflammation. Mol. Med., 5:595-605 (1999). See also Gervais, A., et al. Ex vivo expansion of antitumor cytotoxic lymphocytes with tumor-associated antigen-loaded dendritic cells. Anticancer Research 25, 2177-2185 (2005) and Susskind, B. M. & Chandrasekaran, J. Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine. Journal of Immunology, 139:905-912 (1987).
Szabo and colleagues reported studies exploring the mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase (NOS). See, e.g., Szabo, C., et al. The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites. Br. J. Pharmacol., 113:757-766 (1994).
The NO produced by the enzyme iNOS is a central effector molecule in the innate immune response to pathogens and is the focus of many groups working towards understanding the role of the microbe H. pylori plays in the pathogenesis of stomach ulcers and gastric cancer. Casero and Wilson observed that spermine may inhibit the production of the macrophage-derived NO coming from the inducible NO synthase (iNOS). See, e.g., Bussiere, F. I., et al. Spermine causes loss of innate immune response to Helicobacter pylori by inhibition of inducible nitric-oxide synthase translation. The Journal of Biological Chemistry 280:2409-2412 (2005) and Chaturvedi, R., et al. Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization. The Journal of Biological Chemistry, 279:40161-40173 (2004).
A review article by Soda provides an overview of the immunosuppressive role played by increased polyamine metabolism. See, e.g., Soda, K. The mechanisms by which polyamines accelerate tumor spread. J. Exp. Clin. Cancer Res., 30:95 (2011). However, despite the promise of polyamine pharmaceutical agents, not all reported experiments demonstrate good clinical efficacy for these agents.
N1,N11-diethylnorspermine (DENSpm; DENSPM) was clinically evaluated for therapeutic effect against previously treated metastatic breast cancer, see e.g. Wolff, et al. Clinical Cancer Res., 9:5922-5928 (2003). In this study, DENSpm was delivered as its free base by intravenous infusions over a 15 minute period. Treatment cycles involved injections of 100 mg/m2/day over 5 days every 21 days. A short plasma half-life of 0.5 to 3.7 h was observed. An additional report using DENSpm i.v. infusions for non-small cell lung cancer treatment also failed to demonstrate clinical benefits (Hahm, H. A. et al Clinical Cancer Res., 8:684-690 (2002)).
N1,N14-diethylhomospermine (DEHSpm; DEHSPM) is an additional bis-ethylated polyamine analog tested for clinical efficacy in human oncology trials. Twice daily, subcutaneous injections of this agent as its tetrahydrochloride salt at 12.5, 25 and 37.5 mg/kg in solid tumor patients showed peak drug levels at 15 to 30 minutes after injection. The drug was not observed in plasma of treated patients 2-4 hrs. post-injection (Wilding, G. et al. Investigational New Drugs, 22:131-138 (2004)). None of the 15 patients were found to have an objective response and significant toxicities at the highest dose limited further evaluation in cancer patients.
Squalamine is a chemically synthesized aminosterol, originally isolated from the liver of the dogfish shark. Studies in tumor-bearing mice have shown that squalamine acts as an inhibitor of angiogenesis and shows activity against several models of cancer in mice including lung, breast, ovarian and prostate. Clinical testing of squalamine, as its lactate salt, against advanced non-small cell lung cancer has been reported (Herbst, R. S. Clinical Cancer Res., 9:4108-4115 (2003)). Limited clinical activity was observed in this testing, where squalamine was delivered by continuous i.v. infusions over 3 h at dose levels of 100 to 400 mg/m2/day. Plasma half-life of squalamine was measured to be between 1 and 2 h. An earlier report on the clinical testing of squalamine lactate salt used 120 h continuous i.v. infusion as the delivery method (Bhargava, P. et al. Clinical Cancer Res. 7:3912-3919 (2001)).
Deoxyspergualin is a synthetic analog of the bacteria derived sperqualin and has strong immunomodulatory effects on lymphocytes, macrophages and neutrophils. It is approved for treatment of steroid-resistant transplant rejection in Japan. It is delivered by subcutaneous injections at 0.5 mg/kg/day for up to 21 days. The pharmacokinetic behavior of deoxyspergualin delivery by 3 h intravenous infusions has been reported (Dhingra, K. et al Cancer Research, 55:3060-3067 (1995)) and showed a very short half-life of 1.8 h.
F14512 is a polyamine-epipodophyllotoxin conjugate that is able to target cancer cells with high polyamine transporter activity (Kruczynski, A. et al Leukemia 27:2139-2148 (2013)). It is being developed for use against AML and solid tumors and a recent publication showing its development against canine tumor showed it is delivered by i.v. injections (Tierny, D. Clinical Cancer Res., 21(23):5314-5323 (2015)). Plasma levels of F14512 in dogs treated with 0.05, 0.060, 0.070, 0.075 and 0.085 mg/kg by intravenous 3 hr. infusions increased with dose and were estimated to be within therapeutic range at approximately 2 to 3 hrs. for most dogs.
Mozobil is a bicyclam polyamine-containing drug approved for stem cell mobilization prior to hematopoeitic progenitor cell transplants during cancer chemotherapy (De Clercq, E. Pharmacology and Therapeutics, 128:509-518 (2010)). This drug is administered by subcutaneous injection. Subcutaneous delivery to healthy volunteer patients at 40, 80, 160, 240 and 360 μg/kg showed dose proportional pharmacokinetics and clearance by 10 hrs. Plasma half-life of Mozobil is 3 hrs. (Lack, N. A., et al. Clin. Pharmacol. Ther., 77:427-436 (2005)).
Trientine is a polyamine analog approved for use in Wilson's disease. This polyamine analog acts as a copper chelating agent, aiding in the elimination of excess copper associated with Wilson's disease. Although Trientine is delivered orally, as its hydrochloride salt in the clinic, its oral bioavailability is poor (8 to 30%). It has a relatively short half-life in humans (2 to 4 h). A review covering the preclinical and clinical applications of Trientine has been published. See Lu, J. Triethylenetetramine pharmacology and its clinical applications. Molecular Cancer Therapeutics, 9:2458-2467 (2010).
Methylglyoxal bis(guanylhydrazone), also known as 1,1′[methylethanediylidene]dinitrilodiguanidine and often abbreviated as MGBG, is a polyamine that functions as a competitive polyamine inhibitor of 2-adenosyl methionine decarboxylase (AMD-1), which catalyzes the synthesis of spermidine. It is described as useful in, e.g., the treatment of pain, such as inflammatory pain. See U.S. Pat. Nos. 8,258,186 and 8,609,734.
Oral delivery of spermidine has recently been shown to improve heart health and longevity of mice (Eisenburg, T. et al. Nature Medicine, 22(12):1428-1438 (2016)). Spermidine provided in the diet of mice enhanced cardiac autophagy, mitophagy and mitochondrial respiration and improved the mechano-elastical properties of cardiomyocytes in vivo. The authors attributed the spermidine extension of lifespan of mice to the autophagy inducing activities of spermidine (Eisenburg, T. et al. Autophagy, 13(4):1-3 (2017)).
Lipinski devised a set of parameters that could predict the ability of chemical substances to be orally bioavailable (Lipinski C A, et al. Adv. Drug Deliv. Rev., 46(1-3):3-26 (2001)). Known in the art as ‘The Rule of 5’ these parameters were based on the molecule's chemical structure and included the number of hydrogen bond donors, hydrogen bond acceptors, molecular weight and lipophilicity measurements. Many exceptions to these rules have been found and these parameters are now considered more of a guidance used to predict a molecule's oral bioavailability.
While polyamines have desirable biological properties, the inventor(s) consider that their limited oral bioavailability remains an unsolved hurdle in an effort to bring these materials to practical therapeutic use. In particular, the bioavailability of polyamines by oral administration has been a problem. Surprisingly, oral delivery of polyamine drugs as salts with hydrophobic carboxylic acids greatly improves their bio availabilities. Thus, there exists a need for a pharmaceutical composition that can deliver polyamines and protonated forms thereof to a patient in need, and which overcome one or more of the shortcoming associated with the prior art.
All of the subject matter discussed in the Background section is not necessarily prior art and should not be assumed to be prior art merely as a result of its discussion in the Background section. Along these lines, any recognition of problems in the prior art discussed in the Background section or associated with such subject matter should not be treated as prior art unless expressly stated to be prior art. Instead, the discussion of any subject matter in the Background section should be treated as part of the inventor's approach to the particular problem, which in and of itself may also be inventive.